Integrin and Defensin Modulate the Mechanical Properties ofAdenovirus

Joost Snijder,a Vijay S. Reddy,c Eric R. May,d Wouter H. Roos,a Glen R. Nemerow,b Gijs J. L. Wuitea

Natuur- en Sterrenkunde & LaserLab, VU University, Amsterdam, The Netherlandsa; Department of Immunology and Microbial Science, The Scripps Research Institute, LaJolla, California, USAb; Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USAc; Department of Molecular and Cell Biology, University ofConnecticut, Storrs, Connecticut, USAd

The propensity for capsid disassembly and uncoating of human adenovirus is modulated by interactions with host cell moleculeslike integrins and alpha defensins. Here, we use atomic force microscopy (AFM) nanoindentation to elucidate, at the single-par-ticle level, the mechanism by which binding of these host molecules affects virus particle elasticity. Our results demonstrate thedirect link between integrin or defensin binding and the mechanical properties of the virus. We show that the structure and ge-ometry of adenovirus result in an anisotropic elastic response that relates to icosahedral symmetry. This elastic response changesupon binding host molecules. Whereas integrin binding softens the vertex regions, binding of a human alpha defensin has ex-actly the opposite effect. Our results reveal that the ability of these host molecules to influence adenovirus disassembly cor-relates with a direct effect on the elastic strength of the penton region. Host factors that influence adenovirus infectivitythus modulate the elastic properties of the capsid. Our findings reveal a direct link between virus-host interactions andcapsid mechanics.

Human adenovirus (HAdV) is one of the largest known non-enveloped double-stranded DNA (dsDNA) viruses. The

�36-kb viral genome is encapsidated by a pseudo-T�25 icosahe-dral capsid that is over 90 nm in diameter with a correspondingmass of �150 MDa (1–3). The main capsid proteins form a closedicosahedral shell that is composed of 240 trimeric hexons, 12 pen-tamers of the penton base, and 12 fiber trimers. In addition, thereare four cement capsid proteins (IIIa, VI, VIII, and IX) and fiveproteins associated with the genomic core of the virion (V, VII, �,IVa2, and terminal protein). During the final step of particle mat-uration, multiple capsid proteins are posttranslationally pro-cessed by a viral protease. Approximately one-third of the morethan 50 adenovirus types cause acute infections in humans that aregenerally self-limiting except in immunocompromised patients.Replication-defective adenoviruses are also used in a significantnumber of gene therapy and vaccine applications (4).

Integrin �v�5 is one of several cell surface receptors for ade-novirus that mediates internalization of the virus rather than at-tachment (5–7). Adenovirus binding to integrin �v�5 promotesclustering of the receptors and activates downstream signalingpathways that facilitate virus endocytosis. Integrin binds to ex-posed RGD motifs on the virus penton base in a maximum stoi-chiometry of 4:5 (8, 9). This stoichiometric mismatch betweenintegrin �v�5 and the penton base is the result of a steric hin-drance. It is also believed that integrin binding causes a conforma-tion change in the penton base. The spiral untwisting of the pen-ton base protein due to integrin binding could relax theinteractions of the individual penton base subunits with theneighboring peripentonal hexons as well as cause the release ofthe fiber protein at the cell surface, thereby facilitating capsid dis-assembly at a later stage of cell entry (8, 10).

Adenovirus endosome escape and infection are restricted byhuman alpha defensins. Defensins are a conserved family of anti-microbial proteins, two of which (HNP1 and HD5) are known toblock adenovirus infection (11–14). Binding of defensins to theouter surface of adenovirus blocks endosomal escape and ther-

mally stabilizes the virion. Whereas cell attachment and internal-ization are unaffected by binding of defensins, stabilization of thevirus prevents release of the endosomolytic protein VI and expo-sure of the internal core viral DNA; this prevents subsequent stepsin the virus life cycle, including endosome escape, nuclear local-ization, and ultimately replication of adenovirus in the host cell(15–17). Thus, binding of defensin and integrin to adenovirus hasopposing effects on capsid disassembly and infectivity.

The physical properties of virus capsids are important factorsat various stages of the virus life cycle, including genome packag-ing and capsid maturation (18). In the present study, we deter-mined whether this is also the case for virus-host interactions thatmodulate adenovirus uncoating. While multiple biochemical andstructural analyses also show that integrin and defensin binding toadenovirus produce opposite effects on capsid stability, we testedthis idea more rigorously using a single-particle, mechanobiologi-cal approach. To do this, we used a recently developed tool in thefield of physical virology: atomic force microscopy (AFM) nano-indentation (18, 19). The morphology and mechanical resilienceof the virus capsid can be probed nearly simultaneously in an AFMnanoindentation experiment. The particles are analyzed in buffersolution at ambient temperature. First, an image is recorded, afterwhich nanoindentation is performed on a single-particle basis tomonitor the elastic response of the capsid. Forces can be appliedbeyond a threshold where capsid integrity is maintained, whichallows for the study of mechanical failure of the virion particle.

Received 14 September 2012 Accepted 7 December 2012

Published ahead of print 26 December 2012

Address correspondence to Wouter H. Roos, wroos@few.vu.nl, or Glen R.Nemerow, gnemerow@scripps.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02516-12

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In previous AFM studies of viral mechanics, it was demon-strated that the material properties of virus capsids are intimatelylinked with the viral life cycle. For example, genome encapsidationin cowpea chlorotic mottle virus (CCMV), minute virus of mice(MVM), and phage � was shown to have a profoundly stabilizingeffect on the virus capsid (20–22). The combined process of pro-teolytic maturation and genome packaging in herpes simplex vi-rus 1 nucleocapsids was also shown to increase their mechanicalresilience (23). Similarly, maturation of bacteriophage HK97 alsoresults in capsid stabilization (24). In the HK97 study, it wasshown that covalent cross-linking of capsomeres and reorganiza-tion of the capsid quaternary structure are responsible for theincrease in mechanical strength of the capsid and in its resistanceto material fatigue. Proteolytic maturation has the opposite effectin adenovirus, where the mature capsid is more prone to disas-sembly than are immature capsids, thereby priming the virion forefficient uncoating upon cell entry (25). Finally, phage �29 andnorovirus were shown to have built-in prestress in the capsid thatstrengthens the particles, thereby increasing their survivability inhostile environments (26, 27). Prestress refers to the fact that somecapsid geometries have global nonzero lateral and shear forcesacting on the shell, even in optimal structures at their free-energyminima. While accumulating evidence has demonstrated the im-pact of mechanical stability on the earlier stages of the viral lifecycle, there is still little evidence of its importance in viral infec-tivity. One of the few studies that relate mechanics to infectivitywas performed on a retrovirus, HIV-1, revealing that a “stiffnessswitch” during maturation correlates with the ability of viral par-ticles to enter cells (28).

Here, we present AFM nanoindentation studies of intact hu-man adenovirus type 5 (Ad5) particles equipped with the adeno-virus type 35 (Ad35) fiber, designated Ad35F, to examine the ef-fects of integrin and defensin association. We demonstrate thatbinding of integrin �v�5 and human alpha defensin 5 (�HD5)have opposite effects on the elastic response of Ad35F, revealing adirect link between virus-host interactions and the mechanicalproperties of the capsid.

MATERIALS AND METHODSProteins and virus preparations. The DAV-1 monoclonal antibodythat recognizes the penton base RGD loop was purified on protein G-Sepharose as previously described (29). The purified IgG antibody wasresuspended in 50 mM Tris-HCl, pH 8.0. Soluble recombinant integrin�v�5 was produced in insect cells using baculovirus and purified by im-munoaffinity chromatography as previously described (5). Chemicallysynthesized human alpha defensin HD5 was obtained from Peptides In-ternational, Inc. (Louisville, KY); resuspended at 100 �M in sterile water;and stored at �80°C until use. Human adenovirus type 5 (HAdV5)equipped with the Ad35 fiber, designated Ad35F, was purified as de-scribed previously (1, 30).

AFM. Imaging and nanoindentation were performed at room temper-ature in solution. The buffer for Ad35F–DAV-1 complexes was 40 mMTris (pH 8.1), 500 mM sodium chloride, 2% (wt/vol) sucrose, and 1%(wt/vol) mannitol. The buffer for Ad35F-�v�5 integrin complexes was 20mM Tris (pH 8.1), 150 mM sodium chloride, and 2 mM magnesiumdichloride. The buffer for Ad35F-HD5 complexes was phosphate-buff-ered saline (PBS), 10 mM sodium phosphate (pH 7.4), 137 mM sodiumchloride, 3 mM potassium chloride. The complexes were prepared byincubating either antibody DAV-1, �v�5 integrin, or defensin HD5 withAd35F in a 10-fold molar excess to the penton base monomer for 1 h onice before depositing the solution on the AFM substrate. Free Ad35F was

analyzed under all three buffer conditions. The final Ad35F concentrationduring the measurements was 6 �g/ml.

Free Ad35F and its complexes with either DAV-1, integrin �v�5, ordefensin HD5 were prepared for AFM nanoindentation by incubating a100-�l droplet of virus solution for approximately 15 min at room tem-perature on silanized glass slides. The silanized glass substrates were pre-pared as described previously (19). Briefly, this procedure consisted offirst rinsing the glass slides with deionized water. The slides were driedovernight and then incubated for 16 h in an ethanol-water bath and sat-urated with potassium hydroxide, followed by another round of thoroughrinsing and drying. The cleaned glass slides were rendered hydrophobic byincubating them overnight in a hexamethyldisilazane vapor. After the15-min incubation of the virus solution on the AFM substrate, another100 �l of buffer solution was added before mounting the AFM head witha wetted tip onto the sample. Olympus OMCL-RC800PSA rectangular,silicon-nitride cantilevers with a nominal spring constant of 0.05 N/m anda nominal tip radius of 15 nm were used. The cantilevers were calibratedusing the method of Sader et al. (31), giving an average value of 0.0524 0.002 (standard deviation [SD]) N/m. Viral imaging and nanoindentationwere performed on a Nanotec Electronica AFM (Tres Cantos, Spain),operated in jumping mode. Nanoindentation was performed with a probevelocity of 55 nm/s, and the data were analyzed using a home-built Lab-view application described previously (32). Briefly, the photodiode signalwas converted to units of force based on the slope of a force distance curve(FDC) measured on the glass substrate, where distance is the z-displace-ment of the piezo scanner (see Fig. 1a). The spring constant of the virusparticle was determined from the slope of the FDC on the virus, asdetermined by linear regression on the linear-like part of the FDC,using Hooke’s law, treating the cantilever-virus system as two springsin series. In the case of adenovirus, the linear-like part of the FDCgenerally starts within 0.05 to 0.10 nN of the contact point between thetip and virus and generally ends within 0.05 nN before the breakingpoint. The indentation of the particles at a certain force is determinedby taking the cantilever displacement at the corresponding force of anFDC measured on glass and subtracting this from the cantilever dis-placement of the FDC on the virus particle at the same force. Doingthis for a complete nanoindentation cycle on a particular viral particleyields the force indentation curve (FIC).

The average maximum imaging force was approximately 90 to 120 pN.It was recently reported that AFM imaging of HAdV5 at similar forcesmight result in mechanical failure of the virus (33). We observed damageduring imaging on several occasions (estimated at 10 to 20%) during ourmeasurements, and nanoindentation was not pursued for these particles.For every individual nanoindentation, the integrity of the shell was care-fully checked from the AFM image. Moreover, the relatively high springconstants and breaking forces in our measurements also indicate that themechanical integrity of the shells was preserved during imaging as per-formed prior to nanoindentation. Image processing and analysis wereperformed using WSxM software (34). The tip-sample correction for de-termining the width of force-induced disassembly products was per-formed using simple geometrical considerations, adapted from previouslydescribed procedures (35). The particles were approximated as uprightcylinders with radius r and height h. Then, the broadening half-width, wh,can be estimated as wh � [h1 (2Rt � h1)]0.5 � r, where Rt is the tip radiusand h1 is the height at which wh was determined. For Rt, we used thenominal tip radius of 15 nm, and h1 was set at 0.5h (i.e., the apparent widthwas determined at half the maximum height of the particle, as the fullwidth at half maximum [FWHM]). Thus, at this particular height thefollowing relation holds: FWHM � 2wh.

Normal mode analysis of Mackay icosahedron. To model the effectsof virus nanoindentation, a Mackay icosahedron (36) was constructed,which consisted of 252 nodes and had a radius of �9.5 arbitrary lengthunits (ALU). A nondimension elastic network model was constructed(37), in which any two nodes separated by no more than 4 ALU wereconnected by a harmonic spring. All nodes were given a uniform mass (1

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arbitrary mass unit), and initially all springs were given a uniform stiffness(1 arbitrary force/length unit). This cutoff length permitted connectionsbetween both first-neighbor and second-neighbor nodes; each of the 12nodes situated at 5-fold vertices had 15 connections, while all other nodeshad 17 or 18 connections. From the Hookean potential energy function ofthe elastic network model, a Hessian matrix was constructed and a fulldiagonalization was performed to determine the system’s eigenvalues andeigenvectors (normal modes) of the system. The normal modes are theprincipal directions and provide a natural basis for describing any defor-mation/conformational change. The lowest-frequency modes have beenshown to associate with global/large-scale conformational changes inmany biomolecules and complexes (38). As has been shown previously,for an icosahedrally symmetric structure, all modes were either nonde-generate (unique eigenvalue), indicating icosahedrally symmetric modes,or had 3-, 4-, or 5-fold degeneracy (repeated eigenvalues, same period ofoscillation) (39, 40). We also constructed an elastic network model torepresent �v�5-HAdV by softening the stiffness in the region of the 5-foldvertices. The softening was accomplished by decreasing the spring con-stant of the springs that were directly connected to the nodes at the 5-foldvertices. In order to observe a significant shift in mode frequencies, thesprings connected to the 5-fold node were reduced by a factor of 100. Bysoftening the 5-fold node springs, there is an overall downward shift in theeigenvalue spectrum. Since the overall rigidity of the particles appears toremain intact upon integrin binding, we renormalize the softened net-

work eigenvalues (�), by the ratio��U

��S , where the sums are performed

over the lowest 200 modes, excluding the first 6 modes, which correspondto rigid body movements. With the renormalization, we conserve the sumof the eigenvalues over the low-frequency end of the spectrum betweenthe uniform (U) and softened (S) networks.

Deformations to the model were performed by orienting the modelwith the axis of deformation aligned with the positive Z axis. The defor-mation was performed in spherical coordinates, where the radial posi-tions, ri, were displaced a distance �ri � 2.44cos( i), where i is the polarangle node i makes between the positive Z axis and its radial vector; onlythose atoms in the upper hemisphere of the shell were deformed. Thecosine form of the deformation is analogous to an l1

0 spherical harmonicdeformation, which has previously been shown to be a good descriptor ofthe nanoindentation deformation shape (41). Projections were computedbetween the 200 lowest-frequency normal modes and the displacements,calculated using only the upper hemisphere nodes, by computing normal-ized dot products. The modes were then sorted to identify those modeswhich had the largest-magnitude projection onto the displacements.Those modes which project onto the displacement share components oftheir direction with the direction of the displacement; in essence, those arethe modes which characterize (partially) the motion/displacement of in-terest. We found that the top five modes had nontrivial projections(�0.2), and we used these modes to interpret the stiffness. For a harmonic

oscillator, the frequency of oscillation is given by � �� k

m, where k is the

spring constant and m is the mass of the bead. For a given mode, � � �2,so we can take � � k, and we then sum the �s over the top five modes tocalculate the effective spring constants, which are presented in Results.

RESULTSAFM imaging of intact Ad35F virions. Adenovirus particles(Ad35F) were investigated by AFM on a single-particle basis. First,images were recorded in order to determine the centers of theparticles, the target for the nanoindentation experiments (Fig. 1a).The Ad35F particles average �90 nm in height, which is in agree-ment with previously published cryo-electron microscopy (cryo-EM) and crystal structure data of the Ad35F capsid (Fig. 1b; seealso Table A1 in the Appendix) (1, 2). The icosahedral symmetryof the Ad35F particles can be clearly distinguished from the AFM

FIG 1 AFM imaging of intact Ad35F. (a) Principal components of the AFMsetup. The piezo scanner moves the sample in all directions, allowing for thesharp tip at the end of the cantilever to sample the surface. Topographychanges result in bending of the cantilever and a concomitant change in lasersignal on the quadrant photodiode. The example image of an Ad35F particlelying on a coverslip is shown in three-dimensional rendering under a 77°viewing angle and shows how AFM can accurately image top surfaces but neverbeneath an object. (b) (Top) Surface rendering of Ad35F based on the crystalstructure of the virus (Protein Data Bank [PDB] code, 1VSZ) showing thethree different particle orientations of the icosahedral capsids The four uniquehexon trimers are depicted in red, green, yellow, and blue (peripentonal). Thepenton base is shown in magenta, and the short Ad35 fiber is shown in gold. Asurface-exposed cement protein that forms a helical bundle is depicted in darkblue. (Middle) Ad35F virions imaged by AFM, top-view images. Ad35F has astrongly faceted capsid which can be clearly seen from the AFM images. Typ-ical images of Ad35F deposited on their 2-, 3-, and 5-fold axes are shown asindicated. Each image is 250 by 250 nm in the xy dimension and approximately90 nm in the z dimension. (Bottom) The same images with morphologicalfeatures annotated. Facet edges are indicated by dashed red lines. Several sur-face protrusions can also be distinguished, indicated by white arrowheads, andlikely represent the trimeric fiber. (c) AFM image of free Ad35F with a visiblesurface protrusion on one of the vertices, alongside height profiles along thedirection indicated by the arrows.

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images, both from the hexagonal/pentagonal contour and fromthe triangular facets of the capsid surface (Fig. 1b). One key mor-phological feature of adenovirus capsids is the presence of fibersthat protrude radially outwards from the vertex. The adenovirusstrain of the present study, Ad35F, is based on Ad5, equipped withthe short fiber protein of Ad35. As the fiber is a relatively flexiblecomponent of the capsid (42), it is hard to distinguish this as aclear morphological feature due to the inherent force-applicationof AFM imaging. This is consistent with previous reports on AFMimaging of Ad5, which possesses the longer Ad5 wild-type (WT)fiber, in which no visible fibers were reported (25, 33). In the AFMimages of Ad35F, a small number of particles did exhibit smallsurface protrusions that are located on each of the vertices of theicosahedral shell (Fig. 1b, white arrowheads, and Fig. 1c). Theselikely represent the trimeric fiber of Ad35F. The spikes appearrelatively short compared to the expected length of the Ad35Ffiber protein (expected, �9 nm from the base emerging from thepenton to the tip of the fiber knob; mean height in imaging, 6.6 2.2 nm [SD]; n � 13). On the one hand, this will be accountedfor by the fact that the fiber is unlikely to be perfectly perpen-dicular to the imaging plane and will thus appear shorter basedon simple trigonometric considerations, and on the otherhand, this could indicate that the fibers are partially pushedaway by the AFM tip.

The Ad35F particles are randomly adsorbed onto the substratewith respect to their icosahedral symmetry. As a result, the indi-vidual particles are probed along different icosahedral symmetryaxes, i.e., either on the edge between two triangular facets (2-fold),at the center of a facet (3-fold), or on a vertex (5-fold). Hence, bycorrelating AFM imaging with the nanoindentation results, themechanical response of Ad35F can be distinguished with respectto the icosahedral structure of the capsid. The contact area be-tween the tip and sample is estimated to be on the scale of singlecapsomeres (43). It was recently demonstrated that the elastic re-sponse as probed by AFM correlates with localized structural dy-namics, hence allowing the mechanical response of adenovirus tobe discussed in terms of localized structural dynamics with respectto the icosahedral symmetry of the capsid (44).

Mechanical failure of the virion. Regardless of particle orien-tation, the virions exhibit an initial linear-like force response, andthe spring constant of the particle can be determined from theslope of the FDC. With indentations of �5 to 20 nm, the particlesare no longer able to withstand the relatively high forces, and theysuddenly break (Fig. 2a and 3). This is reflected by sharp transi-tions to lower forces in the curve (Fig. 3a). Imaging of Ad35F afternanoindentation reveals that the high load that was applied to thecapsid results in severe capsid damage (Fig. 2a). Most particlesappear to have completely disintegrated into a large number ofsmaller particles. A subset of particles (5 to 10%) display a holewhere the AFM tip broke through, sometimes accompanied bycracks running along the remainder of the shell. We did not find acorrelation between particle orientation or mechanical responseand the two observed mechanically induced disassembly path-ways.

We further investigated the identity of the force-induced dis-assembly products that appear with the majority of virions. Due totheir large number, they are likely hexon trimers. This was con-firmed by analyzing the height and apparent width of 50 of thesedisassembly products from the AFM images. The hexon trimer asdescribed in the crystal structure of Ad35F is approximately 11.4

nm in height and averages 8.9 nm in width (1) (Fig. 2b). Theapparent height in AFM is not distorted by tip-sample dilationand can be directly extracted from the AFM image (Fig. 2c and d).The 50 analyzed particles average 11.0 0.8 nm in height, whichis consistent with the height of the hexon trimer. The apparentwidth of the particles is heavily dilated by the relatively large tip(nominal tip-radius of 15 nm). The expected, calculated FWHMof the disassembly products is 32 nm as follows from the relationsdescribed in Materials and Methods. However, the measuredFWHM will be lower due to the fact that the hexon will be pushedsideways when imaging off-center and due to the fact that thehexons are not perfect cylinders but rather possess slightly curvedcorners on top. On average, the FWHM of the 50 particles is 25nm, which is a reasonable figure to expect in light of above con-siderations for the hexon trimers (Fig. 2c and d). Hence, the num-ber of observed particles and their dimensions provide major ev-idence that mechanical failure of HAdV results in disassembly intoindividual capsomeres, in particular hexons. It is possible thatsome of these hexons also contain associated cement proteins thatare too small (relative to the hexon) to be detected using thismethod.

Icosahedral symmetry and the mechanical response of thevirion. We observed a rather large spread in the mechanical re-sponse of Ad35F (Fig. 3a). In a two-dimensional analysis of springconstant versus breaking force, three mechanically distinct popu-lations were detected (Fig. 3b). These populations are distin-guished based on the mechanical response according to icosahe-dral symmetry. In particular, the force response of Ad35F issubstantially different when probed on an edge, facet, or vertex(Fig. 3b and c and Table 1). The spring constant and breakingforce are lowest along the 5-fold icosahedral symmetry axis. Thestiffness along the 3-fold symmetry axis is 1.5-fold higher, and thatalong the 2-fold axis is 3.6-fold higher than that on the 5-fold axis.The breaking forces are approximately equal along the 2- and3-fold axes; both are 1.9-fold higher than that on the 5-fold axis.Figure 3c shows all force indentation curves for each of the threeicosahedral axes to illustrate that the mechanical response isstrongly influenced by the icosahedral symmetry of the capsid.Notably, the spring constant obtained along the icosahedral 3-foldsymmetry axis (0.43 N/m) in our studies is in good agreementwith that reported for wild-type Ad5 in an independent studyusing a similar approach (0.46 N/m) (25).

The anisotropic mechanical response of Ad35F, i.e., the differ-ent elastic responses along the three icosahedral axes, can be ex-plained by the structure of the capsid. The anisotropic response oficosahedral shells scales with shell thickness, and the stiffnessalong the 5-fold symmetry axis becomes proportionally lowerwith thicker shells (20). The contiguous part of the Ad35F capsidis approximately 10 nm thick, roughly 25% of the average shellradius. In shells of these dimensions, the force response is ex-pected to be lowest along the 5-fold symmetry axis, consistentwith our results. Around the 2-fold axis of Ad35F, there are severalstructural features that could be related to its relatively high springconstant. First, the hexons at the 2-fold axis have a unique “hand-shake” association comprised of extended hypervariable regionloop associations containing amino residues 184 to 194. This in-teraction is not present between 3-fold related hexons (1). Thecapsid also contains an extensive network of a cement protein (IX)that connects the triangular facets via tetrameric helix bundlesthat are located along the ribs of the shell (1, 2). Figure 4 illustrates

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how the 2-fold axis is reinforced by hexon-hexon and cementprotein associations. The extended conformation of protein VIIImainly stabilizes quasi-3-fold junctions on the capsid interior.However, it also cements the hexons (red) at the 2-fold symmetryaxes with the 3-fold related hexons (green).

Elastic properties of Ad35F in a complex with host cell fac-tors. Next, the effect of integrin �v�5 and defensin HD5 bind-ing to the Ad35F vertex region was investigated. The compo-nents of the buffers used to produce stable virus-host cellmolecular complexes were different from the components ofthe buffer used for the virus alone. Therefore, the mechanicalproperties of free Ad35F were tested in all the different buffersystems used in this study, and no significant effects of buffercomposition were observed (see Table A1 in the Appendix).Ad35F was incubated with an excess of the soluble extracellulardomain of human integrin �v�5 to form a complex and thensubjected to nanoindentation. As with the free particles, integ-rin �v�5-HAdV complexes exhibited an initial linear force re-sponse that was followed by a sudden breaking event (Fig. 5).Differentiating the force response along the different icosahe-

dral symmetry axes revealed that integrin �v�5 binding has asignificant effect on the mechanical response of the capsid(Fig. 6). The stiffness of the vertex region in integrin �v�5-HAdV is 55% lower than that of free particles (P � 0.0001 in aone-way analysis of variance [ANOVA], including the particlesin complex with defensin described below, and P � 0.01 in adirect Bonferroni-corrected two-tailed t test between free andintegrin-bound particles). The spring constant along the 3-foldaxis remains unchanged but is accompanied by a moderate25% increase in the stiffness of the 2-fold axis (P � 0.0007 inthe one-way ANOVA and P � 0.01 in a direct comparisonbetween free and integrin-bound particles in the Bonferroni-corrected two-tailed t test).

To further investigate the seemingly allosteric effects of integ-rin binding, specifically, the increase in stiffness at the 2-fold axisconcomitant with decreased stiffness at the 5-fold vertex, we per-formed a modeling study. The complexity and large size of ade-novirus make it computationally prohibitive for performingstructure-based calculations. Therefore, we performed calcula-tions employing a toy-model, a Mackay icosahedron (36, 40)

FIG 2 AFM imaging of Ad35F reveals virion disassembly upon mechanical failure. (a) Ad35F particles subjected to mechanical failure. The top row shows aparticle that completely disintegrated into individual smaller particles in response to mechanical failure. Left, particle before nanoindentation; right, particle afternanoindentation. The corresponding height profiles are taken along the white arrows indicated in the images. The complete breakage of virions into theircomponent hexon subunits is observed in the vast majority of cases. The bottom row shows a particle that partially maintained capsid integrity upon mechanicalfailure. There is clearly a hole where the AFM tip broke through the shell, as well as a crack running along the remainder of the capsid. This type of response isobserved in approximately 5 to 10% of all particles. Images are 250 by 250 nm in xy and z dimensions as indicated in the corresponding height profile. (b) Surfacerendering of an individual hexon trimer as described in the crystal structure of Ad35F (PDB code, 1VSZ). (c) AFM images of the smaller particles that arise frommechanical failure of Ad35F. The image is 125 by 125 nm in xy dimensions and 15 nm in the z dimension as indicated. (d) A typical height profile of force-induceddisassembly products. A schematic drawing of the hexon trimer is presented to scale, and the imaging parameters that were used to assess the particle dimensionsare indicated on the height profile.

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(Fig. 7a), to investigate if the icosahedral symmetry alone encodesa link between the lower stiffness at the 5-fold axis and greaterstiffness at the 2-fold axis. A simple bead-spring elastic networkmodel was constructed, and the normal modes of the structurewere determined. The normal modes were determined for a net-work with a uniform spring constant (representing free Ad35F)and for a network in which the springs directly connected to thebead at the 5-fold vertex are softened (representing the integrin�v�5-HAdV complex). We then proceeded to deform the capsidalong either the 5-fold or the 2-fold axis (Fig. 7b and c) and deter-

mined which normal modes contribute (project onto) the defor-mation mode. As a control calculation, we also performed thedeformation along the 3-fold axis (not shown).

When the deformation is performed along either the 5-foldor the 2-fold axis, the dominant mode in both the uniform and

FIG 3 AFM nanoindentation of free Ad35F reveals distinct elastic responses along each of the icosahedral symmetry axes. (a) FICs for all individual freeAd35F virions. As the tip approaches and pushes into the virion, there is a steady increase in force. As the indentation increases to between approximately5 and 20 nm, the particles break as revealed by sharp transitions toward lower forces. (b) Two-dimensional density analysis of spring constant versusbreaking force reveals the presence of three distinct populations in the mechanical response of Ad35F. (c) FICs as presented in panel a, binned accordingto the orientation along which the particles were probed. The force response is distinct along each of the three icosahedral symmetry axes, each accountingfor one of the maxima observed in panel b.

TABLE 1 Elastic response of HAdV and virus-host cell molecularcomplexes

Axis Sample

kvira (N/m)

Avg SEM n

2 (edge) Ad35F 1.03 0.04 25Ad35F � integrin 1.25 0.04 15Ad35F � HD5 0.98 0.06 12

3 (facet) Ad35F 0.43 0.01 49Ad35F � integrin 0.41 0.02 23Ad35F � HD5 0.49 0.03 27

5 (vertex) Ad35F 0.31 0.02 36Ad35F � integrin 0.15 0.03 8Ad35F � HD5 0.48 0.04 13

a kvir, virion spring constant.

FIG 4 Surface rendering of Ad35F oriented at its 2-fold axis based on thecrystal structure. The individual proteins are color coded as described for Fig.1. Two lower panels (left and center) show a close-up view of the two hexonsthat are in tight association and in contact with multiple cement proteins.Another lower panel (right) shows a side view of the interacting cement pro-teins that bolster hexon associations.

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softened networks is an icosahedral mode, which is consistentwith the previously observed robustness of icosahedral modesin capsid motions (45, 46). However, there are differenceswhen examining the secondary modes, which still contributesignificantly (projection � 0.2) to the deformation mode. Forthe 5-fold deformation (Fig. 7d) on the softened network, low-frequency modes (a 5-fold degenerate and an icosahedralmode) which are not present in the uniform network contrib-ute to the deformation. Conversely, when the deformation isperformed along the 2-fold axis (Fig. 7e), two higher-frequencymodes (both 3-fold degenerate) contribute to the deformationmode but are not contributing to the deformation on the net-work with uniform springs. By summing the eigenvalues of thetop 5 modes that project onto the deformation mode (thoseshown in Fig. 7d and e), we can estimate an effective springconstant for the deformation. Let us denote these differentspring constants as k5U, k5S, k2U, and k2S, where the U and Ssubscripts represent the uniform or softened network, respec-tively, and the “2” and “5” subscripts represent deformationalong the 5-fold axis or 2-fold axis, respectively. From thisanalysis, we obtain the ratios k5U//k5S � 1.30 and k2U//k2S �0.78.

To further support the applicability of our toy-model to un-derstand elastic properties of virus capsids, we performed the de-formation along a 3-fold axis in both the uniform and softenednetworks. By performing the same analysis as was done for the 2-and 5-fold deformations, we found that the spectrum of eigenval-ues, corresponding to the top modes which project onto the dis-placement, showed little change between the uniform and soft-ened networks. We computed effective spring constants for the3-fold deformation and found the ratio k3U/k3S � 0.98, indicatingthat the stiffness at the 3-fold axis is nearly unchanged upon soft-ening of the 5-fold vertex. These results show that when the spring

constant along the 5-fold axis decreases (due to, for instance, in-tegrin binding), the spring constant along the 2-fold axis increasesand the spring constant along the 3-fold axis is constant. Thesemodeling results qualitatively support the experimental findingson �v�5-HAdV compared to free Ad35F and, given the simplicityof the model, raise the intriguing possibility that these featurescould be observed in other icosahedrally symmetric structures(e.g., other viral capsids).

Next, the mechanical properties of the complex of Ad35Fwith human alpha defensin 5 (�HD5) were studied by nanoin-dentation to reveal whether the virion’s elasticity is affectedupon binding this host cell molecule. We observed a force re-sponse in �HD5-HAdV complexes similar to that in the freevirion (i.e., linear force with indentation followed by suddenbreaking of the shell [Fig. 5]). There was no effect of defensinbinding on the mechanical response of the facets and edges ofthe capsids. However, there was a pronounced increase (66%)in the stiffness of the vertex (P � 0.0001 in a one-way ANOVA,P � 0.001 in the Bonferroni-corrected t test) (Fig. 6). Theseresults show that the ability of alpha defensins to block HAdVdisassembly is correlated with mechanical reinforcement of thepenton region and suggest that defensins prevent release of thepenton via a mechanism that relates to diminished structuraldynamics in this region.

To confirm that the observed effect of integrin �v�5 and de-fensin binding to adenovirus is due to specific interactions withthe penton base, an analysis of adenovirus in complex with the

FIG 5 FDCs of free Ad35F and Ad35F complexed with DAV-1, integrin, andHD5. For each experiment, 10 representative curves are shown in differentcolors. The reference FDC on glass is shown as a dashed black line.

FIG 6 The anisotropic elastic response of Ad35F following host cell molecularinteractions that modulate capsid disassembly. (a) Frequency distributions ofspring constants determined for Ad35F alone, Ad35F-integrin, or Ad35F-HD5complexes. Thin dashed lines indicate the averages determined for Ad35Falone (free); thick dashed lines indicate the averages for Ad35F-host cell mo-lecular complexes. (b) Average spring constants determined for Ad35F,Ad35F-integrin, or Ad35F-HD5 complexes, as determined along each of theicosahedral symmetry axes. Error bars represent standard errors of the means;source data are presented in Table 1.

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monoclonal antibody DAV-1 was included as a control. DAV-1also interacts with the penton base RGD loop, but the intact anti-body has no effect on the infectivity of adenovirus (29). We ob-served no changes in the mechanical response of the DAV-1–Ad35F complex compared to the free particles (Fig. 5; see alsoTable A1 in the Appendix). This confirms that the mechanicalresponses in the integrin �v�5- and defensin-adenovirus com-plexes are uniquely related to their ability to impact both uncoat-ing and infectivity.

DISCUSSION

The mechanical properties of Ad35F were probed with AFMnanoindentation, and we revealed that the elasticity of the cap-sid is correlated with the early events in the virus life cycle, inparticular with virus-host interactions that modulate adenovi-rus capsid disassembly. A striking mechanical feature of Ad35Fis the strong anisotropic response with regard to the icosahe-dral symmetry of the capsid. Integrin �v�5 binding to Ad35Fpentons has been suggested to precede capsid disassembly andrelease of the DNA from the core. Furthermore, it was recentlyshown that the vertex regions of large, faceted viruses are underan internal compressive prestress (47). Due to the prestress atthe 5-fold vertices, the energy penalty of creating a hole forgenome release is likely to be lower at those sites than around it,making the pentons a favorable candidate for a genome releasesite. Here we used a nanoindentation approach to show thatintegrin binding increases the flexibility of the vertex region,

and an increase in flexibility generally precedes structuralevents such as the breaking of protein-protein interactions.The mechanical consequences of a strongly faceted icosahedralshell with a high T number thus appear to promote the infec-tivity of adenovirus through the interaction with its cell surfacereceptor at the 5-fold axis. In contrast, the host defense systemintervenes through the action of defensin HD5, which “ce-ments” the penton into the capsid. This reinforces and stiffensthe vertex of the icosahedral capsid, thus preventing the mostcrucial steps of capsid disassembly and genome uncoating. Theanisotropic mechanical properties of adenovirus alone and inassociation with the host factors thus play a crucial role in theinfectivity of the virus, and several structural features of thecapsid are likely responsible for the anisotropic force response.

The mechanism of how disassembly of the capsid and un-coating of the adenovirus genome are triggered is still underdebate. Cryo-EM data for adenovirus without integrin andcomplexed with integrin indicate that the penton is inserted ina slightly twisted way in the virion and that integrin bindingleads to an untwisting of the penton (8, 9). So, the binding ofintegrin to a penton causes a conformational change relaxingthe interactions of the penton base with the peripentonalhexons. This loosening of the penton base would then facilitateuncoating. In fact, the electron density for the penton baseregion appears to be more diffuse in the complexed particlethan in the free particle, suggesting that a subset of the pentonsmay have already been released from the complex. As it wasrecently demonstrated that the elastic strength of virus capsidscorrelates with localized structural dynamics (44), the currentnanoindentation findings, showing a clear elastic softeningalong the vertex regions, represent a substantial support for thetheory that integrin binding loosens the penton base in theadenovirus capsid. Hence, our AFM nanoindentation experi-ments support the notion that the conformational change inthe penton base of HAdV upon integrin binding facilitates un-coating. Together with the recent results of Pérez-Berná et al.,this indicates that there are at least two consecutive and distinctmechanical events that lead to uncoating (25). The first stepoccurs during assembly and maturation as the capsid organi-zation is loosened by the proteolytic maturation. The secondand final step occurs during infection by complex formation ofthe virions with integrin, leading to the further, necessary de-stabilization of the penton regions and to subsequent genomerelease.

In addition to the large, relative decrease in stiffness alongthe 5-fold symmetry axis, we also observed a moderate increasein stiffness along the 2-fold axis upon integrin binding. This ispossibly related to the localization of stress at the vertices of thecapsid that is the result of the icosahedral structure of the shell.Our modeling results support this notion, by showing that de-formation along a 2-fold axis is characterized by a stiffer effec-tive spring constant, when the network has softer interactionsat the 5-fold vertex, compared to uniform interactionsthroughout. Furthermore, theoretical studies of stress distri-bution in viral capsids show that for high-T-number struc-tures, there is considerable positive lateral and shear stress onthe vertices (48, 49). The stress is apparently relieved from thevertices upon integrin binding: it appears from our compari-son of free Ad35F with the integrin-Ad35F complex that thebuilt-in stress accounts for at least half of the elastic strength of

FIG 7 Normal mode analysis of Mackay icosahedron. (a) Elastic networkrepresentation of the icosahedron. The 5-fold nodes are colored blue; all othernodes are red. (b and c) Deformations along the 5-fold (b) and 2-fold (c) axes.The undeformed configuration is shown by the transparent structure. (d ande) Normal mode projections onto the 5-fold displacement (d) and 2-fold dis-placement (e) are shown against the mode eigenvalues; the 5 modes with thelargest projections are shown. Each mode is identified by its degeneracy (ico-sahedral [I], 5-fold degenerate, or 3-fold degenerate) and whether the networkcontained uniform springs (red circles) or softened springs at the 5-fold ver-tices (blue squares). The modes that are circled in green are the modes mostresponsible for shifting the overall stress response between the uniform andsoftened networks.

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the vertex (it decreases from 0.31 N/m to 0.15 N/m after integ-rin binding).

The mechanism by which HD5 prevents capsid disassemblyand uncoating appears to be quite distinct from how integrinbinding facilitates this process. It was shown in heat-stability as-says of adenovirus that defensin HD5 binding prevents release ofthe penton from the capsid (16). The subsequent release of theinterior capsid protein VI, which is required for endosomal es-cape, is prevented, thus restricting infection (17). Whereas HD5acts on the same crucial step in capsid disassembly as does integ-rin, it does not appear to modulate the stress distribution in theicosahedral shell but simply cements the penton into capsid. Themechanical strength of the vertex region, in terms of the elasticity,is increased by almost 70%, thereby presumably preventing capsiddisassembly and subsequent replication.

Another striking feature in the mechanical response ofAd35F is the complete disassembly of the capsid upon mechan-ical failure. We observed only a small fraction of particleswhere a recognizable capsid remained after nanoindentation,and most particles completely disintegrated into individualcapsomeres. We observed no evidence for preservation ofGroup-Of-Nine/Six hexons, indicating that force-induced dis-assembly is more complete than that achieved by treatmentswith organic solvents such as pyridine. Markedly different ef-fects of mechanical failure on capsid integrity have been re-ported for other virus capsids. In herpes simplex virus 1, theAFM tip was shown to penetrate the capsid upon mechanicalfailure, displacing several capsomeres but leaving the rest of theshell mostly intact (23). A similar pattern of just several dis-placed capsomeres was reported for phage �29 (35). Phage �and HK97 proheads also exhibit discrete fracture patterns (21,24). In hepatitis B virus and HK97 head II, it was shown thatcapsid integrity is almost completely maintained upon me-

chanical failure, apart from the observation that capsid heightis not fully restored (24, 32, 50). Even though clear differencesin mechanical failure can be distinguished between differentvirus capsids, a common theme for adenovirus, herpes simplexvirus 1, and �29 is also apparent. In all three systems, it is evidentthat mechanically induced disassembly follows a path that is de-lineated by penton-hexon or hexon-hexon boundaries. This con-firms that these capsomeres are fundamental structural units ofvirus (dis)assembly.

In conclusion, we demonstrated with a direct experimentalapproach that virus-host cell molecular interactions impact themechanical properties of the virus capsid. Whereas previousmechanical studies focused on the overall stability of virionparticles, we were able to reveal the actual location and mech-anism of stabilization and destabilization. Host cell moleculeswith opposite effects on capsid disassembly modulate the elas-tic response of the HAdV virion accordingly. The infectivity ofHAdV appears crucially dependent on the icosahedral geome-try of the capsid, mainly through the anisotropic stiffness andstress distribution that arise as a consequence of specific struc-tural features of the virion. These findings represent significantadvances in our understanding of viral infectivity, as we haveuncovered a direct link between virus-host interactions thatcritically influence cell entry processes and the mechanicalproperties of virus capsids.

APPENDIX

The complexes of Ad35F with DAV-1, integrin, or defensin were incu-bated and measured by AFM in different buffer solutions. Hence, to en-sure that none of the observed effects of host-factor binding on the elasticresponse were due to the accompanying differences in buffer composi-tion, we performed control experiments on free Ad35F in all correspond-

TABLE A1 Summary of Ad35F nanoindentationa

Axis Preparation Buffer

k (N/m) Fbreak (nN) h (nm)

nAvg SEM Avg SEM Avg SEM

2 Ad35F DX 1.03 0.04 5.4 0.3 91.3 0.6 10TMN 1.00 0.07 4.0 0.2 92.4 0.5 10PBS 1.03 0.08 4.5 0.2 91.4 0.4 5

Ad35F � DAV-1 DX 0.96 0.06 4.8 0.3 93.1 0.3 10Ad35F � integrin TMN 1.25 0.04 5.3 0.3 92.2 0.3 15Ad35F � HD5 PBS 0.98 0.06 5.4 0.2 92.6 0.6 12

3 Ad35F DX 0.42 0.03 4.6 0.2 91.5 0.3 21TMN 0.44 0.02 4.6 0.2 92.1 0.3 20PBS 0.41 0.02 4.2 0.3 92.1 0.3 8

Ad35F � DAV-1 DX 0.40 0.04 4.9 0.2 91.2 0.5 8Ad35F � integrin TMN 0.41 0.02 5.1 0.2 92.9 0.3 23Ad35F � HD5 PBS 0.49 0.03 4.2 0.2 91.5 0.2 27

5 Ad35F DX 0.29 0.01 3.6 0.5 95.4 0.6 10TMN 0.33 0.03 2.4 0.2 93.7 0.4 16PBS 0.29 0.04 2.0 0.4 94.5 0.5 10

Ad35F � DAV-1 DX 0.27 0.04 2.7 0.4 93.8 0.7 9Ad35F � integrin TMN 0.15 0.03 2.4 0.3 95.4 0.6 8Ad35F � HD5 PBS 0.48 0.04 3.6 0.5 94.8 0.3 13

a Reported are the average virion spring constant k, breaking force Fbreak, and maximum particle height h. Buffer conditions as follows: DX, 40 mM Tris (pH 8.1), 500 mM sodiumchloride, 2% (wt/vol) sucrose, and 1% (wt/vol) mannitol; TMN, 20 mM Tris (pH 8.1), 150 mM sodium chloride, and 2 mM magnesium dichloride; PBS, 10 mM sodium phosphate(pH 7.4), 137 mM sodium chloride, and 3 mM potassium chloride.

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ing buffers. Table A1 lists these results together with the average heightand breaking forces found in our experiments.

ACKNOWLEDGMENTS

G.J.L.W. is supported by an STW-administered NanoSci E� grant, byFundamenteel Onderzoek der Materie (FOM) through the “Physics of thegenome” program, and by the NanoNextNL consortium of the DutchGovernment and partners. G.R.N. received support from NIH grants RO1HL054352 and EY011431. V.S.R. received support from NIH grantAI070771.

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